The levels of cytokines IL-6, IL-8, and INFγ were analyzed using a Human Cytokine A Premixed Magnetic Luminex Performance Assay (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Cytokine concentrations were determined using the dual laser flow analyzer Luminex 100/200 (Luminex Corp., Austin, TX, USA). Standard curves for each cytokine were plotted using a 5-parameter logistic fit (5-PL). Each sample was run in duplicate. BEAS-2B was exposed at 25 μg/mL of PM cocktail (of SEQB-B and SEQB-H), and pro-inflammatory cytokines IL-6 and IL-8 were quantified, together with INFγ, to explore inflammation as a response to 4 and 8 h exposure with (0.01% DMSO in BEBM medium was used as controls).
Human cytokine a premixed magnetic luminex performance assay
Manufactured by R&D Systems
Sourced in United States
The Human Cytokine A Premixed Magnetic Luminex Performance Assay is a multiplex immunoassay designed to measure multiple analytes simultaneously in a single sample. The assay utilizes color-coded magnetic beads coated with antibodies specific to the target analytes. Upon incubation with the sample, the analytes of interest bind to their respective antibodies. Detection is achieved through the use of a biotinylated detection antibody and streptavidin-phycoerythrin conjugate, which generates a fluorescent signal proportional to the amount of each analyte present in the sample.
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3 protocols using human cytokine a premixed magnetic luminex performance assay
1
Cytokine Response to PM Exposure
2
Biomarkers for Inflammatory Processes
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CRP, MIC-1, and sTNF-RII were assayed in the laboratory of Dr. Nader Rifai (Boston Children’s Hospital, Boston, MA). The sTNF-RII is an established surrogate measurement for TNF-α due to its role in TNF-α signaling, lower diurnal variation, and increased stability in frozen plasma25 (link),26 (link). Furthermore, unlike TNF-α levels of which tend to fluctuate, levels of sTNF-RII are stable over long periods of time27 (link). CRP was measured using an immunoturbidimetric assay (Roche Diagnostics, Indianapolis, IN), with a limit of detection of 0.03 mg/L. MIC-1 and sTNF-RII were measured by an ELISA assay (R&D Systems, Minneapolis, MN), with a sensitivity of 4.36 pg/mL for MIC-1 and 0.6 pg/mL for sTNF-RII. IL-6 was measured as part of the 16-plex pro- and anti-inflammatory cytokine panel (Human Cytokine A Premixed Magnetic Luminex Performance Assay, R&D Systems, Minneapolis, MN). The sensitivity of the assay is 1.11 pg/mL. Coefficients of variation for each assay were calculated using 10% blinded duplicate samples, and ranged from 2.5% for CRP to 6.1% for sTNF-RII. Laboratory personnel was blinded to patient status.
3
Multiplex ELISA for Cytokine and Antibody Quantification
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The concentrations of cytokines and chemokines were determined in NHP serum by a magnetic-based multiplex ELISA 26 analytes (NHP XL Cytokine Luminex Performance Premixed Panel, R&D systems or Human cytokine A Premixed Magnetic Luminex Performance Assay, R&D systems, for IL18 and IL1-RA), following the manufacturer’s instructions. The concentrations of anti-VSV.G Abs were determined in NHP serum by ELISA, coating with recombinant VSV.G (Alpha Diagnostic Intl.) 1 μg/mL and developing with a HRP conjugated mouse anti-monkey IgG Ab (Southern Biotech 1:10,000).
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